Journal: Journal of medicinal chemistry

Article Title: Development of a Redox-Sensitive Spermine Prodrug for the Potential Treatment of Snyder Robinson Syndrome

doi: 10.1021/acs.jmedchem.1c00419

Figure Lengend Snippet: Polyamine levels in wild-type (a: CMS-24949) and SMS mutant (b: CMS-26559, c: CMS-6233, and d: CMS-23916) fibroblast cell lines with or without treatment of Spm or prodrug 1 (5 μM) in the presence of AG (1 mM) after 72 h incubation at 37 °C. Values represent data from the experiment performed in triplicates ± SD, *p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: The authors wish to thank Dr. Charles Schwartz at the Greenwood Genetic Center for supplying the SRS cell lines and control wt fibroblasts and the Snyder Robinson Foundation for financial support of this work (UCF25068A04 to O.P.).

Techniques: Mutagenesis, Incubation

Journal: Journal of medicinal chemistry

Article Title: Development of a Redox-Sensitive Spermine Prodrug for the Potential Treatment of Snyder Robinson Syndrome

doi: 10.1021/acs.jmedchem.1c00419

Figure Lengend Snippet: Polyamine levels in CMS-23916 (mutant) fibroblast cell lines untreated (UT) or treated with prodrug 1 (5 μM) only, NAC (2 mM) only, or a combination of prodrug 1 (5 μM) and NAC (2 mM). Values represent data from the experiment performed in triplicates ± S.D. *p < 0.05.

Article Snippet: The authors wish to thank Dr. Charles Schwartz at the Greenwood Genetic Center for supplying the SRS cell lines and control wt fibroblasts and the Snyder Robinson Foundation for financial support of this work (UCF25068A04 to O.P.).

Techniques: Mutagenesis

Journal: Cell reports

Article Title: Suppressing Aneuploidy-Associated Phenotypes Improves the Fitness of Trisomy 21 Cells

doi: 10.1016/j.celrep.2019.10.059

Figure Lengend Snippet: (A) Representative images of RPE-1 nuclei mock treated and treated with myriocin for 24 h. Immunofluorescence for lamin B1 is red, and the nucleus is blue stained with Hoechst 33342. (B) Percentage of nuclear abnormalities of RPE-1 cells treated with myriocin (n = 100 cells). (C) Representative images of RPE-1 nuclei upon knockdown of SPTLC1 or SPTLC2 after 72 h. (D) Percentage of nuclear abnormalities of RPE-1 cells upon knockdown of SPTLC1 or SPTLC2 (n = 100 cells). (E) Western blot analysis of SPTLC1 and SPTLC2 protein levels in RPE-1 cells. (F) Representative images of RPE-1 nuclei treated with DHS, sphingosine (Sph), sphingosine-1-phosphate (S1P), or ceramide (Cer) for 24 h. (G) Percentage of RPE-1 cells showing abnormal nuclear morphology when treated with DHS, sphingosine (Sph), sphingosine-1-phosphate (S1P), or ceramide (n = 100 cells). (H) Representative images of primary human skin fibroblast (HSF) nuclei mock treated and treated with myriocin for 24 h. (I) Percentage of nuclear abnormalities of primary HSF treated with myriocin (n = 100 cells). (J) Representative images of primary HSF nuclei treated with DHS, sphingosine (Sph), sphingosine-1-phosphate (S1P), or ceramide for 24 h. (K) Percentage of primary HSF showing abnormal nuclear morphology when treated with DHS, sphingosine (Sph), sphingosine-1-phosphate (S1P), or ceramide (Cer) (n = 100 cells). All images are representative of three biological replicates. Scale bars (A, C, F, H, and J), 10 μm.

Article Snippet: Euploid human skin fibroblast (Control)_2 , Coriell Institute , GM08447; RRID:CVCL_7487.

Techniques: Immunofluorescence, Staining, Knockdown, Western Blot

Journal: Cell reports

Article Title: Suppressing Aneuploidy-Associated Phenotypes Improves the Fitness of Trisomy 21 Cells

doi: 10.1016/j.celrep.2019.10.059

Figure Lengend Snippet: (A) Representative images of primary human fibroblasts from 2 euploid donors and 2 patients with Down syndrome. Immunofluorescence for lamin B1 is red, and the nucleus is blue stained with Hoechst 33342. Scale bar, 2.5 μm. (B) Percentage of fibroblasts from euploid donors showing abnormal nuclear morphology when treated with fumonisin B 1 or SKi-II (n = 100). (C) Percentage of fibroblasts from 2 patients with Down syndrome showing abnormal nuclear morphology treated with fumonisin B 1 , SKi-II, or ceramide (Cer) (n = 100). (D) Representative images of trisomy 21 nuclei treated with fumonisin B 1 , SKi-II, or ceramide. Scale bar, 10 μm. (E) Representative images of primary human fibroblasts from 2 euploid donors and 2 patients with Patau syndrome(trisomy 13) or Edward syndrome (trisomy 18). Immunofluorescence for lamin B1 is red, and the nucleus is blue stained with Hoechst 33342. Scale bar, 2.5 μm. (F) Percentage of fibroblasts from 2 patients with Patau syndrome showing abnormal nuclear morphology treated with fumonisin B1, SKi-II, orceramide (n = 100). (G) Percentage of fibroblasts from 2 patients with Edward syndrome showing abnormal nuclear morphology treated with fumonisin B1, Ski-II, or ceramide (n = 100).

Article Snippet: Euploid human skin fibroblast (Control)_2 , Coriell Institute , GM08447; RRID:CVCL_7487.

Techniques: Immunofluorescence, Staining

Journal: Cell reports

Article Title: Suppressing Aneuploidy-Associated Phenotypes Improves the Fitness of Trisomy 21 Cells

doi: 10.1016/j.celrep.2019.10.059

Figure Lengend Snippet: (A) Representative images of primary human fibroblasts from euploid donors and patients with Patau syndrome (trisomy 13), Edward syndrome (trisomy 18), or Down syndrome (trisomy 21). Immunofluorescence is shown for lamin A/C, H3K9triMe, and Hoechst 33342. In the merge images, lamin A/C is green, H3K9triMe is red, and DNA is blue. Scale bar, 5 μm. (B) Western blot analysis of H3K9triMe in human fibroblasts. (C) Quantification of the number of 53BP1 foci in human fibroblasts mock treated or in the presence of fumonisin B 1 (n = 100). (D) Representative images of primary human fibroblasts from euploid donors and patients with Down syndrome. Immunofluorescence is shown for 53BP1 (red) and Hoechst 33342 (blue). There is no correlation between abnormal nuclear morphology and number of 53BP1 foci. Scale bar, 5 μm. (E) Growth curves of primary human fibroblasts from 2 euploid donors and 2 patients with Patau (trisomy13), Edward (trisomy18), or Down syndrome(trisomy 21) with increasing concentrations of fumonisin B 1 . (F) Representative images of primary human astrocytes from 4 euploid donors and 4 donors with Down syndrome. Immunofluorescence for the nucleus is blue stained with Hoechst 33342. Scale bar, 5 μm. (G) Percentage of astrocytes from euploid and trisomy 21 donors showing abnormal nuclear morphology (n > 200).

Article Snippet: Euploid human skin fibroblast (Control)_2 , Coriell Institute , GM08447; RRID:CVCL_7487.

Techniques: Immunofluorescence, Western Blot, Staining

Journal: Cell reports

Article Title: Suppressing Aneuploidy-Associated Phenotypes Improves the Fitness of Trisomy 21 Cells

doi: 10.1016/j.celrep.2019.10.059

Figure Lengend Snippet:

Article Snippet: Euploid human skin fibroblast (Control)_2 , Coriell Institute , GM08447; RRID:CVCL_7487.

Techniques: Recombinant, Membrane, Protease Inhibitor, Control, Software

Journal: Scientific Reports

Article Title: Identification of p38 MAPK as a novel therapeutic target for Friedreich’s ataxia

doi: 10.1038/s41598-018-23168-x

Figure Lengend Snippet: Partial loss of phenotype in seed-sequence variant of gFA11A. ( a ) Primary FRDA fibroblasts GM3816 were transfected with gFA11, Mut1, and Mut2 siRNAs (Fig. ). The cells were transfected twice over 7 days. Cells were kept in DMEM + 5 mM BHB after the first transfection. **p < 0.01 by ANOVA with Tukey pair-wise comparisons. ( b ) Primary FRDA fibroblasts GM3816 were transfected with gFA11, Mut1, or control (C3) siRNA, or mock transfected, every 3–4 days for a total of four times over 14 days. Cells were kept in DMEM + 5 mM glucose throughout. **p < 0.01 and ***p < 0.005 by ANOVA with Tukey pair-wise comparisons. Error bars represent means ± 1 SD. The averages shown were calculated on three independent replicates and the results are representative of at least two independent experiments.

Article Snippet: We obtained primary FRDA fibroblasts, GM3816 and GM3665B; age- and sex-matched apparently healthy control fibroblasts, GM8400 and GM8399; and FRDA lymphocytes, 15850, from Coriell (Coriell Institute for Medical Research, Camden, NJ).

Techniques: Sequencing, Variant Assay, Transfection, Control

Journal: Scientific Reports

Article Title: Identification of p38 MAPK as a novel therapeutic target for Friedreich’s ataxia

doi: 10.1038/s41598-018-23168-x

Figure Lengend Snippet: Alterations in cytokine secretion induced by gFA11. ( a ) Primary FRDA GM3816 fibroblasts were transfected in triplicate with gFA11 siRNA or Mut1 siRNA four times over twelve days. After the first transfection, the cells were grown in DMEM plus 5 mM BHB. At day 12, after the fourth transfection, cells were incubated with DMEM with BHB but without FBS. The following day, the medium was collected, concentrated, and analyzed by Luminex assay. Cytokine concentrations were normalized by cell numbers. ( b ) Primary FRDA GM3816 fibroblasts were infected with a gFA11-encoding vector or empty vector (E). Infected cells were selected with puromycin and expanded in glucose-based medium. Cells were then seeded at low density and kept in medium without FBS for 24 hours before collecting, concentrating, and analyzing the medium. *p < 0.05; **p < 0.01; ***p < 0.005 by Student’s t test. Error bars represent means ± 1 SD. The averages shown were calculated on three ( a ) or four ( b ) independent replicates and the results are representative of two independent experiments.

Article Snippet: We obtained primary FRDA fibroblasts, GM3816 and GM3665B; age- and sex-matched apparently healthy control fibroblasts, GM8400 and GM8399; and FRDA lymphocytes, 15850, from Coriell (Coriell Institute for Medical Research, Camden, NJ).

Techniques: Transfection, Incubation, Luminex, Infection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Identification of p38 MAPK as a novel therapeutic target for Friedreich’s ataxia

doi: 10.1038/s41598-018-23168-x

Figure Lengend Snippet: Effects of gFA11 on cell cycle and morphology. ( a ) Morphology of primary FRDA GM3816 fibroblasts transfected with gFA11 siRNA or Mut1 siRNA, or not transfected. Arrows indicate senescent-appearing cells. ( b ) Flow-cytometric side scattering (SSC) of GM3816 and GM3665B cells infected with a gFA11-encoding or control-encoding (C3) vector. ***p < 0.005 by Student’s t test. ( c ) Cell-cycle analysis of GM3816 cells, untransfected or transfected 4 times with gFA11 siRNA over two weeks. Error bars represent means ± 1 SD.

Article Snippet: We obtained primary FRDA fibroblasts, GM3816 and GM3665B; age- and sex-matched apparently healthy control fibroblasts, GM8400 and GM8399; and FRDA lymphocytes, 15850, from Coriell (Coriell Institute for Medical Research, Camden, NJ).

Techniques: Transfection, Infection, Control, Plasmid Preparation, Cell Cycle Assay

Journal: Scientific Reports

Article Title: Identification of p38 MAPK as a novel therapeutic target for Friedreich’s ataxia

doi: 10.1038/s41598-018-23168-x

Figure Lengend Snippet: Activation of p38 MAP kinase in FRDA cells. ( a ) Frataxin protein levels (by ELISA) in normal control fibroblasts (6030) and primary FRDA fibroblasts (4675, 156, 4491, 203). ***p < 0.005 by ANOVA with Tukey pair-wise comparisons. (The aggregate p value comparing the normal control to the FRDA cells combined was less than 1 × 10 -8 ). ( b ) p38 phosphorylation in the same cells as in ( a ) *p < 0.05 by ANOVA with Tukey pair-wise comparisons. (156 and 4491 cells grew extremely slowly and only two replicates were possible; the aggregate p value comparing the normal control to the FRDA cells combined was 0.008). ( c ) Frataxin protein levels (by ELISA) in normal control fibroblasts (6030) transfected with a random siRNA, C3 (siC3), a known toxic siRNA, C5 (siC5), or an siRNA to frataxin (siFXN) after one transfection (left bar) or two transfections (right bar). The decrease with siFXN was associated with p < 0.005 in both cases by Student’s t test. ( d ) p38 phosphorylation in the same cells as in ( c ) *p < 0.05; ***p < 0.005 by Student’s t test. Error bars represent means ± 1 SD. The averages shown were calculated on three independent replicates and the results are representative of at least two independent experiments.

Article Snippet: We obtained primary FRDA fibroblasts, GM3816 and GM3665B; age- and sex-matched apparently healthy control fibroblasts, GM8400 and GM8399; and FRDA lymphocytes, 15850, from Coriell (Coriell Institute for Medical Research, Camden, NJ).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Control, Transfection

Journal: Scientific Reports

Article Title: Identification of p38 MAPK as a novel therapeutic target for Friedreich’s ataxia

doi: 10.1038/s41598-018-23168-x

Figure Lengend Snippet: p38 inhibitors increase the growth of FRDA cells in a dose-dependent manner. ( a ) p38 phosphorylation status in control GM8399 fibroblasts and FRDA GM3816 fibroblasts. ***p < 0.005 by Student’s t test. ( b ) Primary FRDA GM3816 fibroblasts treated every 48 h with the p38 inhibitor BIRB796 at the concentrations indicated. Cells were counted at day 14. **p < 0.01 relative to carrier control (CC) by ANOVA with Tukey pair-wise comparisons. (100 nM reached a p value of 0.051 relative to carrier control.) Error bars represent means ± 1 SD. ( c ) Primary FRDA GM3816 fibroblasts treated every 48 h with the p38 inhibitor BIRB796, at the concentrations indicated. Cells were counted at day 7. (d) Primary DL156 primary FRDA fibroblasts were treated every 48 h with the p38 inhibitor BIRB796, at a concentration of 500 nM. *p < 0.05 relative to carrier control (CC) by Student’s t test. **p < 0.01; ***p < 0.005; relative to carrier control (CC) by ANOVA with Tukey pair-wise comparisons. Error bars represent means ± 1 SD. In all experiments, the averages shown were calculated on three independent replicates and the results are representative of at least two independent experiments.

Article Snippet: We obtained primary FRDA fibroblasts, GM3816 and GM3665B; age- and sex-matched apparently healthy control fibroblasts, GM8400 and GM8399; and FRDA lymphocytes, 15850, from Coriell (Coriell Institute for Medical Research, Camden, NJ).

Techniques: Control, Concentration Assay

Journal: Scientific Reports

Article Title: Identification of p38 MAPK as a novel therapeutic target for Friedreich’s ataxia

doi: 10.1038/s41598-018-23168-x

Figure Lengend Snippet: p38 phosphorylation is not sustained by cytokines. Primary apparently healthy fibroblasts GM8399 were transfected with FXN siRNA or a random control clone. Two hours after transfection, cells were incubated with DMEM without FBS in presence of 500 nM BIRB796 or carrier control. The following day, ( a ) frataxin protein levels, ( b ) p38 phosphorylation levels, and ( c ) IL-6 levels were measured by ELISA. **p < 0.01; ***p < 0.005 by ANOVA with Tukey pair-wise comparisons. Error bars represent means ± 1 SD. In all experiments, the averages shown were calculated on three independent replicates and the results are representative of at least two independent experiments.

Article Snippet: We obtained primary FRDA fibroblasts, GM3816 and GM3665B; age- and sex-matched apparently healthy control fibroblasts, GM8400 and GM8399; and FRDA lymphocytes, 15850, from Coriell (Coriell Institute for Medical Research, Camden, NJ).

Techniques: Transfection, Control, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Dissecting the molecular organization of the translocon-associated protein complex

doi: 10.1038/ncomms14516

Figure Lengend Snippet: ( a ) Western blot analysis of control and TRAPδ-deficient patient primary fibroblasts. HeLa cells were used as positive control for the TRAP antibodies. Please note that bands for HeLa cells and control fibroblasts are duplicated with because samples were run on the same gel. ( b ) Subtomogram average of the ER-associated ribosome from TRAPδ-deficient primary fibroblasts, including the large (blue) and small (yellow) ribosomal subunits, the membrane bilayer (grey), the Sec61 protein-conducting channel (dark blue), OST (red) and TRAP (green). A total of 2319 subtomograms were averaged. Scale bar, 10 nm. ( c ) Zoomed view of the area indicated in b . The difference density map (magenta) between the TRAPδ-deficient and wild-type mammalian translocons is superimposed on the subtomogram average (grey). ( d ) Structure of the wild-type mammalian translocon (black mesh) superposed with the elements shown in c . TRAP and OST are shown from the perspective of the ER lumen, with the membrane removed. The contact site between TRAP and OST in the wild-type mammalian translocon is indicated (red circle).

Article Snippet: TRAPγ-deficient cells (CDG359), TRAPδ-deficient cells (CDG406) and control fibroblasts (GM0038, from the Coriell Institute) were cultured in DMEM 1 g per L glucose with L -glutamine and sodium pyruvate (HyClone, GE Healthcare, Freiburg, Germany) containing 10% FBS (Sigma-Aldrich, Taufkirchen, Germany) and 1% penicillin and streptomycin (Gibco, Thermo Fisher Scientific, Darmstadt, Germany) in a humidified environment with 5% CO 2 at 37 °C.

Techniques: Western Blot, Control, Positive Control, Membrane

Journal: Nature Communications

Article Title: Dissecting the molecular organization of the translocon-associated protein complex

doi: 10.1038/ncomms14516

Figure Lengend Snippet: Relative protein content of TRAP subunits and select ER-resident proteins in patient fibroblasts.

Article Snippet: TRAPγ-deficient cells (CDG359), TRAPδ-deficient cells (CDG406) and control fibroblasts (GM0038, from the Coriell Institute) were cultured in DMEM 1 g per L glucose with L -glutamine and sodium pyruvate (HyClone, GE Healthcare, Freiburg, Germany) containing 10% FBS (Sigma-Aldrich, Taufkirchen, Germany) and 1% penicillin and streptomycin (Gibco, Thermo Fisher Scientific, Darmstadt, Germany) in a humidified environment with 5% CO 2 at 37 °C.

Techniques: Membrane

Journal: Nature Communications

Article Title: Dissecting the molecular organization of the translocon-associated protein complex

doi: 10.1038/ncomms14516

Figure Lengend Snippet: ( a ) Western blot analysis of control and TRAPγ-deficient patient primary fibroblasts. HeLa cells were used as positive control for the TRAP antibodies. Please note that bands for HeLa cells and control fibroblasts are duplicated with because samples were run on the same gel. ( b ) Subtomogram average of the ER-associated ribosome from TRAPγ-deficient primary fibroblasts, including the large (blue) and small (yellow) ribosomal subunits, the membrane bilayer (grey), the Sec61 protein-conducting channel (dark blue) and OST (red). A total of 663 subtomograms were averaged. Scale bar, 10 nm. ( c ) The difference density map (magenta) between the TRAPγ-deficient and wild-type mammalian translocons is superimposed on the subtomogram average (grey).

Article Snippet: TRAPγ-deficient cells (CDG359), TRAPδ-deficient cells (CDG406) and control fibroblasts (GM0038, from the Coriell Institute) were cultured in DMEM 1 g per L glucose with L -glutamine and sodium pyruvate (HyClone, GE Healthcare, Freiburg, Germany) containing 10% FBS (Sigma-Aldrich, Taufkirchen, Germany) and 1% penicillin and streptomycin (Gibco, Thermo Fisher Scientific, Darmstadt, Germany) in a humidified environment with 5% CO 2 at 37 °C.

Techniques: Western Blot, Control, Positive Control, Membrane

Journal: Nature Communications

Article Title: Dissecting the molecular organization of the translocon-associated protein complex

doi: 10.1038/ncomms14516

Figure Lengend Snippet: ( a ) Set of difference densities (DD) originating from the TRAPδ-deficient fibroblast translocon (blue mesh) and the algal translocon (red mesh) mapped back on the isolated density of the TRAP complex from EMD-3068 (green). For the lumenal TRAP segment, difference densities originating from the TRAPδ-deficient fibroblast translocon and the algal translocon co-localize (asterisks). Scale bar, 2 nm. ( b ) Approximate positions of TRAPγ (red outline), TRAPδ (blue outline) and the TRAPα–β heterodimer (magenta outline), assigned according to the set of difference densities in a . The path of a nascent protein through the Sec61 complex (dashed red line) was traced based on PDB 5EUL. View and colouring matches .

Article Snippet: TRAPγ-deficient cells (CDG359), TRAPδ-deficient cells (CDG406) and control fibroblasts (GM0038, from the Coriell Institute) were cultured in DMEM 1 g per L glucose with L -glutamine and sodium pyruvate (HyClone, GE Healthcare, Freiburg, Germany) containing 10% FBS (Sigma-Aldrich, Taufkirchen, Germany) and 1% penicillin and streptomycin (Gibco, Thermo Fisher Scientific, Darmstadt, Germany) in a humidified environment with 5% CO 2 at 37 °C.

Techniques: Isolation